Recombinant lymphotoxin cDNA and variants

ABSTRACT

Biologically active lymphotoxin polypeptides are synthesized in recombinant cell culture. Novel nucleic acid and vectors incorporating same are provided. The compositions and processes herein enable the economical preparation of compositions containing uniform lymphotoxin polypeptides and variant lymphotoxins having amino acid sequences that differ from those found in nature. The lymphotoxins are purified to a specific activity of 2-10×10 7  units/mg of protein by purification using a novel immobilized, lymphotoxin-neutralizing monoclonal antibody.

This application is a divisional of application Ser. No. 07/836,765filed on 14 Feb. 1992, now U.S. Pat. No. 5,683,688, which is adivisional application of Ser. No. 06/732,312 filed on 9 May 1985, nowabandoned, which is a continuation-in-part application of Ser. No.06/616,503, filed May 31, 1984, now abandoned.

Reference is made to related copending U.S. Ser. No. 608,316, filed May7, 1984, entitled "Human Lymphotoxin" and U.S. Ser. No. 616,502, filedMay 31, 1984, entitled "Anti-Lymphotoxin".

BACKGROUND

This application relates to lymphokines. In particular, it relates tolymphotoxin and derivatives thereof.

Lymphotoxin was first identified as a biological factor withanticellular activity on neoplastic cell lines. An activity identifiedas lymphotoxin and obtained from mitogen-stimulated lymphocytes isassociated with a spectrum of cytotoxic activities ranging fromcytostasis of certain tumor cell lines to marked cytolysis of othertransformed cells. However, lymphotoxin activity is characterized bylittle or no anticellular activity on primary cell cultures and normalcell lines tested. This putative discriminating anticellular property oflymphotoxin led to in vivo studies which suggest that lymphotoxin mayhave a potent antitumor activity.

Lymphotoxin is the term applied to what has been described as a familyof molecules. Lymphotoxin molecules have been identified asglycoproteins divided into five molecular weight classes, each of whichin turn is heterogenous with respect to charge. The human alpha (MW70-90,000) and beta (MW 25-50,000) classes appear to predominate in mostlymphocyte supernatants. The alpha MW classes can be separated by chargeinto at least seven subclasses, while the beta subclass has beenseparated into two distinct subclasses (G. Granger et al. in Mozes etal., Ed., 1981, Cellular Responses to Molecular Modulators pp 287-310).Also identified have been complex (MW >200,000) and gamma (MW 10-20,000)lymphotoxin forms. The various lymphotoxin forms and classes differ fromone another in their stability and kinetics of appearance in culture.Furthermore, they may aggregate together with the complex class underconditions of low ionic strength. The lower molecular weight classes oflymphotoxins have been disclosed to be relatively unstable and weaklycell lytic compared to the higher molecular weight classes (Hiserodt etal., 1976, "Cell. Immun." 26: 211; Granger et al. in De Weck et al. Ed.,1980 Biochemical Characterization of Lymphokines pp 279-283). Gammaclass activity has not been studied extensively because of itsinstability (G. Granger et al., 1978 "Cellular Immunology" 38: 388-402).The beta class also has been reported to be unstable (Walker et al., "J.of Immun." 116 3!: 807-815 March 1976!).

It should be understood that lymphokine terminology is not uniform. Atpresent, the names given to cell culture products are largely a functionof the cells which are believed to elaborate the product and theperformance of the products in biological assays. However, theseproducts remain poorly characterized in large measure because manystudies have been conducted with partially pure preparations and becausethe assays used to characterize the products are not molecule-specificand in any case are subject to considerable variation. The true identityof the various cytotoxic factors will remain unknown in the absence ofstandard terminology based on clearly assayable distinguishingcharacteristics such as amino acid sequences or immune epitopes. Asexamples of other names given to cytotoxic cell culture products aretumor necrosis factor, NK cell cytotoxic factor, hemorrhagic necrosisfactor and macrophage cytotoxin or cytotoxic factor.

Copending and commonly assigned U.S. Ser. No. 06/608,316, filed May 7,1984, now abandoned and EP 100,641A (published Feb. 15, 1984) describeamino acid sequences for a human lymphotoxin isolated from the humanlymphoblastoid cell line RPMI-1788. The continuation of U.S. Ser. No.06/608,316 has now issued as U.S. Pat. No. 4,920,196.

Hayashi et al., EP 132,125A (published Jan. 23, 1985) describerecovering a protein from a rabbit following stimulation of itsreticuloendothelial system. The protein was reported to have antitumoractivity and the N-terminal amino acid sequenceSer-Ala-Ser-Arg-Ala-Leu-Ser-Asp-Lys-Pro-Leu-Ala-His-Val-Val-Ala-Asn-Pro-Gln-Val-Glu-Gly-Gln-Seu-Gln-Trp-Leu.

Copending and commonly assigned U.S. Ser. No. 628,059, filed Jul. 5,1984, now abdandoned discloses the purification and recombinantsynthesis of a cytotoxic human polypeptide identified as tumor necrosisfactor and having the N-terminal amino acid sequenceVal-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp-Lys-Pro-Val-Ala-His-Val-Val-Ala-Asn-Pro.

Ohnishi et al. (U.S. Pat. No. 4,481,137) discloses obtaining a 7-9,000MW substance named CB_(x3) from BALL-1 cell culture that suppresses thegrowth of tumor cells and that has an Ala-Ala N-terminus.

According to Toth and Granger, "Mol. Immun." 16: 671-679 (1979), neitherthe removal of sialic acid from lymphotoxin-containing lymphocytesupernatants by neuraminodase treatment nor the addition ofN-acetyl-glucosamine, galactose, lactose, mannose, α-methyl-mannoside orfucose to the supernatants had any affect on in vitro lytic activity.Toth et al. thus concluded that simple sugars do not appear to play arole in the activity of their lymphotoxin. However, Toth et al. alsoobserve that saccharides play an important role in the action of otherlymphokines and concluded that they could not exclude the participationof more complicated forms of oligo saccharides in the cytotoxic activityof lymphotoxin.

Subsequently, Proctor, Klostergaard and Granger ("Clinical Research",1982, 30(1): 55A) reported that human lymphocytes, when activated by PHAin the presence of tunicamycin (to inhibit the addition of N-linkedcarbohydrate moieties to lymphotoxin molecules), released biologicallyinert lymphotoxin. According to these authors, immunochemical studiesrevealed that while the carbohydrate moiety of lymphotoxin was notneeded for its transport and release by the activated lymphocyte intothe supernatant, the carbohydrate was needed in order to have effectivetarget cell destruction because the carbohydrate was responsible for theappropriate conformation of the lymphotoxin molecule(s).

Other literature that should be studied in connection with thisapplication includes Evans, "Cancer Immunol. Immunother." 12: 181-190(1982); Lee et al., "Cell. Immun." 48: 166-181 (1979); De Weck et al.Ed., (1980) Biochemical Characterization of Lymphokines pp 279-312; Khanet al. Ed. (Jun. 30, 1982) Human Lymphokines pp 459-477; Aggarwal etal., Presentation at the 3rd International Lymphokine workshop inHaverford, Pa., Aug. 1-5 1982; Ransom et al., "Cancer Research" 43:5222-5227 (November 1983); Kull et al., "J. of Immun." 126(4): 1279-1283(April 1981); J. Sawada, et al., "Jpn. J. Exp. Med." 46: 263-267 (1976);G. Granger et al., "Cell. Immunol." 38: 388-402 (1978); J. Rundell etal., "Immunopharmacology" 3: 9-18 (1981); G. Granger et al., "J.Lymphokine Res." 1: 45-49 (1982); N. Ruddle et al., "Lymphokine Res." 2:23-31 (1983); M. Mitsuhashi et al., U.K. Patent Application 2,106,117;H. Enomoto, European Patent Application 87,087A; B. Williamson et al.,"P.N.A.S. USA" 80:5397-5401 (1983) and S. Wright et al., "J. Immunol."126: 1516-1521 (1981).

The lymphotoxin (or substances identified as lymphotoxin) obtainedheretofore from lymphocyte culture are present in low concentrations, onthe order of 0.05-2×10⁶ units/l in supernatants of RPMI-1788 cells orprimary lymphocytes. The amounts harvested often vary considerably, andprimary lymphocytes are expensive. An economical method for producinglymphotoxin is needed (Yamamoto et al., "J. of Biological ResponseModifiers" 3: 1! 76-87 1984!).

Prior methods also fail to produce lymphotoxin which is homogeneous asto amino acid sequence, an important feature for drug utilities.Lymphotoxin recovered from cell line culture exhibits amino terminalheterogeneity, probably due to proteolytic processing (see the abovecited U.S. Pat. No. 4,920,196. Cultures of primary lymphocytes, e.g.from adenoids or peripheral blood, must necessarily contain the cells ofmany donors for reasons of economy. However, the products of these cellswill reflect genetic variation among the donors so that the resulting"lymphotoxin" may in fact be a mixture of allelic species. Obviously,the proportions and identities of such alleles will be unknown fromlot-to-lot. A method is needed for producing lymphotoxin that is uniformas to its amino acid sequence.

Prior methods also are limited to the production of lymphotoxin havingprimary amino acid sequences corresponding to those found in nature.Substituting, deleting or inserting different amino acids in thesesequences would require extensive and costly chemical modifications, ifsuch could be accomplished at all. Methods are needed for easilyintroducing variations into the amino acid sequences of lymphotoxin.

Although the antitumor effects and apparent therapeutic value oflymphotoxin activity have been reported in the literature since 1968,lymphotoxin has not been studied in extensive clinical protocols orcommercialized due to the small quantities and heterogenous nature ofthe lymphotoxin made available through prior methods. Methods are neededto economically prepare quantities of lymphotoxin adequate for clinicalstudies.

Rabbit antisera have been described in the literature which are capableof neutralizing the cytolytic activity of various cytotoxins, includingsubstances identified as lymphotoxin (Yamamoto et al. "Cell. Immun." 38:403-416 (1978); Gately et al., "Cell. Immun." 27: 82-93 (1976); Hiserodtet al., "J. of Immun." 119(2): 374-380 (1977); Zacharchuk et al.,"P.N.A.S. USA" 80: 6341-6345 (October 1983); Ruddle et al., "LymphokineResearch" 2(1) 23-31 (1983); Mannel et al., "Infection and Immunity"33(1): 156-164 (1981); Wallach et al. E. De Maeyer et al. Ed. TheBiology of the Interferon System pp 293-302 (Pub. September 1983) andStone-Wolff et al., "J. Exp. Med." 159: 828-843 (March 1984). Sincethese antisera are polyclonal it contains a multiplicity of antibodiesdirected against the immunogen lymphotoxin. Any one or more of theseantibodies is acting to neutralize the "lymphotoxin" activity. Further,the literature reports generally are unclear as to the molecularidentity of the substance responsible for lymphotoxin activity that wasused as the immunogen. What is needed for diagnosis and immunoaffinitypurification procedures is a monospecific antibody directed against aclearly and unambiguously identified lymphotoxin molecule. It is anobjective of this invention to provide such an antibody.

It is a further object herein to provide methods for economicallysynthesizing a lymphotoxin form in a composition wherein the primaryamino acid sequence of substantially all of the lymphotoxin molecules isthe same.

It is another object to produce predetermined variations in the aminoacid sequence of a lymphotoxin form, more specifically, amino aciddeletions, insertions, substitutions, or combinations thereof.

SUMMARY OF THE INVENTION

The objectives of this invention have been accomplished by thesuccessful recombinant expression of protein having lymphotoxinactivity. This lymphotoxin species, which is described herein in termsof its activity and natural or variant amino acid sequence, ishenceforth referred to as lymphotoxin. Surprisingly, the DNA encodinglymphotoxin has been identified notwithstanding the minute levels oflymphotoxin expressed in homologous cells and uncertainty as to the timeat which messenger RNA encoding lymphotoxin appears in homologous cells.Also surprisingly, biologically active lymphotoxin is expressed inrecombinant cells that do not glycosylate the lymphotoxin (or that wouldnot be expected to do so in the same fashion as homologous cells) andthe lymphotoxin so expressed is recovered having a substantially uniformamino acid sequence, without N-terminal enzymatic hydrolysis. DNAencoding lymphotoxin is expressed in cell cultures in copious quantitiesexceeding 0.1 to 1×10¹¹ units/liter of culture lysate.

The lymphotoxin that is expressed by a recombinant host cell will dependupon the DNA employed to encode the lymphotoxin or its precursors aswell as upon the host cell selected. The nucleic acid sequences employedherein for lymphotoxin synthesis are novel. They are characterized bynucleotide sequences that differ from the native or natural sequence inone or more of the following ways: The DNA is free of introns, in thecase of human lymphotoxin the intron present between nucleotides 284 and285 (FIG. 2a); the DNA is free of nucleic acid encoding other proteinsof the organism from which the DNA originated; the nucleic acid encodinglymphotoxin is ligated into a vector; and/or the nucleic acid is capableof hybridizing to nucleic acid encoding lymphotoxin provided, however,that such hybridizing nucleic acid does not have the nucleotide sequenceof natural DNA or RNA encoding lymphotoxin.

Mutant nucleic acids encoding lymphotoxin are the product of recombinantmanipulations. Silent mutations in the 5' untranslated or translatednucleic acid for lymphotoxin are-provided in order to enhance expressionlevels in selected hosts, e.g. by reducing the probability ofstem-and-loop messenger RNA structures in the 5' regions of the nucleicacid, or by substituting host-preferred codons for those found innatural nucleic acid isolates.

Mutations in the nucleic acids which are expressed rather than silentenable the preparation of lymphotoxin species having the amino acidsequence of native lymphotoxin or primary sequence variants thereof withamino acid sequences differing from the native lymphotoxin. The mutantlymphotoxin is recovered as such or is further processed by the hostcell to obtain the desired lymphotoxin species.

These nucleic acids or nucleic acids that hybridize therewith, orfragments thereof, are labelled and used in hybridization assays for theidentification or determination of genetic material encodinglymphotoxin.

In processes for the synthesis of lymphotoxin, DNA which encodeslymphotoxin is ligated into a vector, the vector used to transform hostcells, the host cells cultured and lymphotoxin recovered from theculture. This general process is used to synthesize lymphotoxin havingthe amino acid sequence of native lymphotoxin or to construct novellymphotoxin variants, depending upon vector construction and the hostcell chosen for transformation. The lymphotoxin species which arecapable of synthesis herein include leucyl amino-terminal lymphotoxin,histidyl amino-terminal lymphotoxin, pre lymphotoxin, and lymphotoxinvariants including (a) fusion proteins wherein a heterologous protein orpolypeptide is linked by a peptide bond to the amino and/orcarboxyl-terminal amino acids of lymphotoxin, (b) lymphotoxin fragments,especially fragments of pre lymphotoxin in which any amino acid between-34 and +23 is the amino-terminal amino acid of the fragment, (c)lymphotoxin mutants wherein one or more amino acid residues aresubstituted, inserted or deleted, (d) methionyl or modified methionyl(such as formyl methionyl or other blocked methionyl species)amino-terminal derivatives, and/or (e) unglycosylated or variantlyglycosylated species of all of the foregoing.

If a mammalian cell is transformed with nucleic acid encodinglymphotoxin operably ligated to a eucaryotic secretory leader (includingthe native lymphotoxin secretory leader), or if nucleic acid whichencodes lymphotoxin is operably ligated in a vector to a procaryotic oryeast secretory leader which is recognized by the host cell to betransformed (usually the organism from which the leader sequence wasobtained), the host transformed with the vector and cultured, thennonmethionylated amino terminal lymphotoxin species ordinarily arerecovered from the culture.

If DNA encoding lymphotoxin is operably ligated into a vector without asecretory leader sequence and then used to transform a host cell, thelymphotoxin species which are synthesized are generally substituted withan amino-terminal methionyl or modified methionyl residue such as formylmethionyl.

Methods are provided whereby in vitro mutagenesis of the nucleic acidencoding lymphotoxin leads to the expression of lymphotoxin variants notheretofore available. First, N-terminal methionyl or modified methionyllymphotoxin is expressed by host cells transformed with nucleic acidencoding lymphotoxin which is directly expressed, i.e., which is notoperably linked to a secretory leader sequence.

Secondly, in vitro, site-specific, predetermined or random mutagenesisis employed to introduce deletions, substitutions and/or insertions intothe nucleic acid that encodes lymphotoxin. Lymphotoxin fusions areproduced in this manner. The lymphotoxin derivatives obtained uponexpression of mutant nucleic acid exhibit modified characteristics.

Finally, unglycosylated or variantly glycosylated lymphotoxins areprovided as novel lymphotoxin species. Unglycosylated lymphotoxin isproduced by prokaryotic expression of DNA encoding lymphotoxin.Variantly glycosylated lymphotoxin species are the product ofrecombinant culture in transformed higher eukaryotic, ordinarilymammalian, cells.

The lymphotoxin produced herein is purified from culture supernatants orlysates by immunoaffinity adsorption using insolubilizedlymphotoxin-neutralizing antibody. This antibody, which is mostefficiently produced in monoclonal cell culture, is raised in mice byimmunization with alum-adsorbed lymphotoxin.

The lymphotoxin of this invention is combined for therapeutic use withphysiologically innocuous stabilizers and excipients and prepared insterile dosage form as by lyophilization in dosage vials or storage instabilized aqueous preparations. Alternatively, the lymphotoxin isincorporated into a polymer matrix for implantation into tumors orsurgical sites from which tumors have been excised, thereby effecting atimed-release of the lymphotoxin in a localized high gradientconcentration.

The therapeutic compositions herein are administered in therapeuticallyeffective doses by implantation, injection or infusion into animals,particularly human patients, that bear malignant tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a depicts a DNA sequence and its putative amino acid sequenceencoding a lymphotoxin fragment.

FIG. 1b demonstrates the construction of synthetic DNA encoding the FIG.1a fragment.

FIG. 2a shows the complete amino acid sequence for pre lymphotoxin, itscoding DNA plus 5' and 3' flanking untranslated regions.

FIG. 2b illustrates a method of constructing an expression vector formethionyl leucyl amino-terminal lymphotoxin and its amino terminalmethionyl derivatives.

FIG. 3 shows a method of constructing an expression vector for methionylhistidyl amino-terminal lymphotoxin.

FIG. 4 depicts the amino acid sequences for human, murine and bovinelymphotoxin, and the consensus mammalian lymphotoxin residues.

FIGS. 5a and 5b depict the construction of a plasmid encoding a fusionof lymphotoxin and a bacterial signal sequence.

DETAILED DESCRIPTION

Lymphotoxin is defined for the purposes of this application as abiologically active polypeptide having a region demonstratingsubstantial structural amino acid homology with at least a portion ofthe lymphotoxin amino acid sequence shown in FIG. 2a. Biologicalactivity is defined as preferential, cytotoxic activity as definedbelow, immunological cross-reactivity with a cytotoxic lymphotoxin orthe ability to compete with cytotoxic lymphotoxin for lymphotoxin cellsurface receptors. In the latter two instances the lymphotoxin need notbe cytotoxic per se. Immunologically cross-reactive mutants are usefulas immunogens for raising anti-lymphotoxin in animals, e.g. for thepreparation of immunoassay reagents, while non-cytotoxic competitivemutants find utility as labelled reagents in competitive-typeimmunoassays for biologically active lymphotoxin.

Preferential cytotoxic activity is defined as the preferentialdestruction or growth inhibition of tumor cells in vivo or in vitro whencompared to normal cells under the same conditions. Destruction of tumorcells by lysis in vitro or necrosis in vivo is the preferred assayendpoint, although cytostasis or antiproliferative activity also is usedsatisfactorily.

Suitable assays for detecting the anticellular activities of lymphotoxinare described in B. Aggarwal, et al., 1984, "J. Biol. Chem." 259 (1),686-691 and E. Carswell, et al., 1975, "Proc. Natl. Acad. Sci. USA" 72,3666-3670.

Lymphotoxin specific activity is defined herein in terms of target celllysis, rather than cytostasis. One unit of lymphotoxin is defined as theamount required for 50 percent lysis of target cells plated in each wellas is further described in Example 1. However, other methods fordetermining cytotoxic activity are acceptable.

Substantial structural homology generally means that greater than about60 percent, and usually greater than about 70 percent of the amino acidresidues in the polypeptide are the same or conservative substitutionsfor the corresponding residue(s) in the sequence of FIG. 2a.

Not all of the sequence of a lymphotoxin polypeptide need be homologouswith the FIG. 2a sequence. Only a portion thereof need be homologouswith any portion of the FIG. 2a sequence so long as the candidateexhibits the required biological activity. Generally, homology should bedemonstrable for regions of about from 20 to 100 amino acid residues,recognizing that occasional gaps may need to be introduced in order tomaximize the homology.

Less homology is required for polypeptides to fall within the definitionif the region of homology with the FIG. 2a sequence is not in one of thelymphotoxin key regions, i.e. regions that are important for cytotoxicactivity. The key regions of the FIG. 2a sequence are believed to beabout residues 162-171, 52-83 and 127-148.

Lymphotoxin is defined to specifically exclude human tumor necrosisfactor or its natural animal analogues (D. Pennica et al., "Nature"312:20/27 December, 1984, pp. 724-729 and B. Aggarwal et al., "J. Biol.Chem." 260 4!: 2345-2354 1985!).

Structurally similar refers to dominant characteristics of the aminoacid side chains such as basic, neutral or acid, hydrophilic orhydrophobic, or the presence or absence of steric bulk. Substitution ofone structurally similar amino acid for another generally is known inthe art as a conservative substitution.

A significant factor in establishing the identity of a polypeptide aslymphotoxin is the ability of antisera which are capable ofsubstantially neutralizing the cytolytic activity of substantiallyhomogeneous, lymphoblastoid (or natural) lymphotoxin to alsosubstantially neutralize the cytolytic activity of the polypeptide inquestion. However it will be recognized that immunological identity andcytotoxic identity are not necessarily coextensive. A neutralizingantibody for the lymphotoxin of FIG. 2a may not bind a candidate proteinbecause the neutralizing antibody happens to be directed to a site onlymphotoxin that merely neighbors a region that is critical tolymphotoxin cytotoxic activity, but which acts as a neutralizingantibody by steric hinderance of the lymphotoxin active site. Acandidate protein mutated in this innocuous region might no longer bindthe neutralizing antibody, but it would nonetheless be lymphotoxin interms of substantial homology and biological activity.

Lymphotoxin obtained by culture of lymphoblastoid cell lines has beendetermined to have the following characteristics: A molecular weight of20,000 or 25,000, depending upon the degree of glycosylation andN-terminal heterogeneity; glycosylation at Asn+62 (FIG. 2a); a tendencyto aggregate, particularly to organize into multimers; an isoelectricpoint of about 5.8; pH lability (a loss of >50 percent of cytolyticactivity when stored for 24 hours in ammonium bicarbonate buffer at 10μg/ml concentration with pH levels less than about 5 or greater thanabout 10); and substantial losses in activity upon incubation in aqueoussolution for 5 min. at 80° C. Two lymphoblastoid lymphotoxin molecularweight species have been identified. The 25,000 da species oflymphoblastoid lymphotoxin has an amino-terminal leucine residue.Polypeptides having the amino acid sequence of the 25,000 da species arecalled leucyl amino-terminal lymphotoxin. The 20,000 da species oflymphoblastoid lymphotoxin is characterized by an amino-terminalhistidine and corresponding sequences are termed histidyl amino-terminallymphotoxin. It is important to observe that these characteristicsdescribe the native or wild type human lymphotoxin obtained fromlymphoblastoid cell cultures. While lymphotoxin as defined hereinincludes native, glycosylated lymphotoxin, other related cytotoxicpolypeptides many fall within the scope of the definition. For example,the glycosylation ordinarily associated with an animal lymphotoxin maybe modified upon expression in a heterologous recombinant eukaryotichost cell, thereby bringing the modified lymphotoxin outside of themolecular weights or isoelectric point established for humanlymphoblastoid lymphotoxin. Lymphotoxin which is entirely unglycosylatedis produced in recombinant bacterial culture with its molecular weight,isoelectric point and other characteristics correspondingly modified. Inaddition, post-translational processing of pre lymphotoxin from a firstanimal species in a cell line derived from another animal species mayresult in a different amino-terminal residue than is ordinarily the casefor the first animal species. Similarly, the mutagenesis proceduresprovided herein, for example, will enable one to vary the amino acidsequence and N-terminus of lymphotoxin, thereby modifying the pHstability, isoelectric point and the like.

The translated amino acid sequence for human lymphotoxin is described inFIG. 2a. Note that this sequence includes a 34 residue presequence whichis believed to be removed during normal processing of the translatedtranscript in human cells (herein, together with its mutants, "prelymphotoxin"), resulting in the leucyl amino terminal species. Thehistidyl amino-terminal species is homologous to the leucylamino-terminal species except that the first 23 amino acids of theleucyl amino-terminal species are absent. All three species, i.e. prelymphotoxin, leucyl amino-terminal lymphotoxin and histidylamino-terminal lymphotoxin, as well as their methionyl, modifiedmethionyl, mutant and unglycosylated forms, are included within thescope of lymphotoxin. The unglycosylated leucyl and histidylamino-terminal species will have lower molecular weights than describedabove for the homologous species from lymphoblastoid cells.

Pre lymphotoxin is a species of lymphotoxin included within theforegoing definition. It is characterized by the presence of a signal(or leader) polypeptide at the amino terminus of the molecule.Generally, the native signal polypeptide of lymphotoxin isproteolytically cleaved from lymphotoxin as part of the secretoryprocess in which the protein is secreted from the cell. The signalpeptide may be microbial or mammalian (including the native, 34 residuepresequence), but it preferably is a signal which is homologous to thehost cell. Some signal-lymphotoxin fusions are not recognized or"processed" by the host cell into N-terminal met-free lymphotoxin. Suchfusions containing microbial signals have utility for example aslymphotoxin immunogens.

Note that the language "capable" of cytotoxic activity means thatlymphotoxin includes polypeptides which can be converted, as byenzymatic hydrolysis, from an inactive state analogous to a zymogen to apolypeptide fragment which exhibits the desired biological activity. Thelanguage "capable" of in vitro or in vivo cytotoxic activity is intendedto embrace noncytotoxic polypeptides which can be converted, as byenzymatic hydrolysis, from an inactive state analogous to a zymogen to apolypeptide fragment which exhibits the definitional biologicalactivity. Typically, inactive precursors will be fusion proteins inwhich lymphotoxin is linked by a peptide bond at its carboxyl terminusto another protein or polypeptide. The sequence at this peptide bond ornearby is selected, so as to be susceptible to proteolytic hydrolysis torelease lymphotoxin, either in vivo or, as part of a manufacturingprotocol, in vitro. Typical linking sequences are lys-lys or arg-lys.The nonlymphotoxin component to such a prolymphotoxin is preferably ahomologous protein so as to minimize the immunogenicity of the fusion.The homologous protein should be innocuous and not bind to cellsurfaces. The lymphotoxin that is so generated then will exhibit thedefinitionally-required cytotoxic activity.

While lymphotoxin ordinarily means human lymphotoxin, lymphotoxin fromsources such as murine, porcine, equine or bovine is included within thedefinition of lymphotoxin so long as it otherwise meets the standardsdescribed above for homologous regions and biological activity. Forexample, bovine and murine lymphotoxins have been found to be highly(about 80 percent) homologous with human lymphotoxin. Lymphotoxin is notspecies specific, e.g., human lymphotoxin is active on mouse tumors andneoplastic cell lines. Therefore, lymphotoxin from one species can beused in therapy of another.

Lymphotoxin also includes multimeric forms. Lymphotoxin spontaneouslyaggregates into multimers, usually dimers or higher multimers. Multimersare cytotoxic and accordingly are suitable for use in in vivo therapy.Lymphotoxin is expressed in recombinant hosts as a monomer. However,lymphotoxin thereafter tends to spontaneously form multimers.Homogeneous multimers or a mixture of different multimers aretherapeutically useful.

Variant lymphotoxins include predetermined or targeted, i.e. sitespecific, mutations of the FIG. 2a molecule or its fragments. Variantlymphotoxins are defined as polypeptides otherwise meeting the definedcharacteristics of lymphotoxin but which are characterized by an aminoacid sequence that differs from that of FIG. 2a, whether by omission,substitution or insertion of residues. The nonhuman lymphotoxinsdescribed herein, and alleles of human lymphotoxin, are to be consideredvariant lymphotoxins, as are site-directed mutants having no naturalcounterpart. The objective of mutagenesis is to construct DNA thatencodes lymphotoxin as defined above but exhibits characteristics thatmodify the biological activity of natural lymphotoxin or facilitate themanufacture of lymphotoxin. For example, the lysine +89 codon is mutatedin order to express a histidine residue in place of the lysine residue.The histidine +89 is no longer hydrolyzed by trypsin (which generallycleaves proteins at an arg-X or lys-X bond). Protease resistance isexpected to confer greater biological half life on the mutant than isthe case for lymphotoxin having the sequence of FIG. 2a (or a fragmentthereof). Other lymphotoxin lysine or arginine residues may be mutatedto histidine, for example lysine +28, lysine +19 or arginine +15.

As discussed above, certain regions of the lymphotoxin molecule exhibitsubstantial homology with a similarly-active protein designated tumornecrosis factor. Amino acid residues in and immediately flankingthese-substantially homologous regions are preferred for mutagenesisdirected to identifying lymphotoxin mutants that exhibit variantbiological or cytotoxic activity. Such mutants are made by methods knownper se and then screened for the desired biological activity, e.g.increased cytotoxicity towards the particular neoplasm being treated or,in the case of lymphotoxin species intended for immunization of animals,the ability to elicit a more potent Immune response. Examples of suchlymphotoxin. variants are as follows: Ala+168 is mutated to a branchedchain amino acid (val, ile, or leu); a hydrophobic amino acid (e.g.,phe, val, ile or leu) is inserted between thr+163 and val+164; tyrosinesubstituted for thr+163; lysine substituted for ser+82; isoleucine,leucine, phenylalanine, valine or histidine substituted for ser+42;glutamine, tryptophan, serine or histidine substituted for lys+84;ser+82 deleted; a hydrophobic di-or tripeptide fused to leu+171;aspartic acid or lysine substituted for thr+163; ala-lys insertedbetween glu+127 and pro+128; lysine or glycine substituted for ser+70;tyrosine substituted for thr+69; arginine or histidine substituted forlys+28; arginine or lysine substituted for his+32; proline, serine,threonine, tyrosine or glutamic acid substituted for asp+36; tyrosine,methionine or glutamic acid substituted for ser+38; threonine, tyrosine,histidine, or lysine substituted for ser+61; aspartic acid, serine ortyrosine substituted for gly+124; arginine, lysine, tyrosine, tryptophanor proline substituted for his+135; aspartic acid substituted forthr+142; and lysine or threonine substituted for gln+146.

A particularly desirable group of mutants are those in which themethionine residues at human lymphotoxin residues +20, +120 and +133 aredeleted or, preferably, substituted for by the corresponding residuesfound in the lymphotoxins of other species such as are describedelsewhere herein. For example met+20, +120 and +133 are substituted bythreonine, serine and valine, respectively. These are the correspondingresidues in bovine lymphotoxin. The substitution is effected in themanner described in Example 9 except that met+133 is mutated to val by afurther step of mutagenesis using M13 Mp8 phage in accord with methodsknown per se. This mutant animal species-hybrid lymphotoxin DNA is usedin place of the leucyl amino-terminal DNA of Example 7 and expressed asa fusion. Following known procedures, cyanogen bromide is used to cleavethe STII signal from the hybrid lymphotoxin and the mature leucylamino-terminal lymphotoxin variant recovered.

Other useful variant lymphotoxins are those in which residues from tumornecrosis factor are substituted for corresponding lymphotoxin residuesto produce hybrid tumor necrosis factor-lymphotoxin variants. Arepresentative example is the substitution of the first 8, 9 or 10residues of mature tumor necrosis factor (e.g.,val-arg-ser-ser-ser-arg-thr-pro-ser-asp-) for the first 27 residues ofleucyl amino-terminal lymphotoxin. This variant is more likely to beN-terminal demethionylated upon direct expression in E. coli.

While the mutation site is predetermined, it is unnecessary that themutation per se be predetermined. For example, in order to optimize theperformance of the mutant histidine +89 lymphotoxin, random mutagenesisis conducted at the codon for lysine +89 and the expressed lymphotoxinmutants screened for the optimal combination of cytotoxic activity andprotease resistance.

Lymphotoxin also may contain insertions, usually on the order of aboutfrom 1 to 10 amino acid residues, or deletions of about from 1 to 30residues. Substitutions, deletions, insertions or any subcombination maybe combined to arrive at a final construct. Insertions include amino orcarboxyl-terminal fusions, e.g. a hydrophobic extension added to thecarboxyl terminus. Preferably, however, only substitution mutagenesis isconducted. Obviously, the mutations in the encoding DNA must not placethe sequence out of reading frame and preferably will not createcomplementary regions that could produce secondary mRNA structure.Extracts of E. coli transformed with vectors containing DNA encodinglymphotoxin mutants having a deletion of the last 16 carboxy terminalamino acids or deletion of the first about 33 amino terminal residues ofleucyl amino-terminal lymphotoxin exhibited no cytotoxic activity.However, the reasons for lack of activity are not known and could havebeen any of those set forth in Example 1 infra.

Not all mutations in the DNA which encodes the lymphotoxin will beexpressed in the ultimated product of recombinant cell culture. Forexample, a major class of DNA substitution mutations are those DNAs inwhich a different secretory leader has been substituted for the FIG. 2asecretory leader, either by deletions within the 34 residue leader or bysubstitutions, which exchange of most or all of the native leader for aleader more likely to be recognized by the intended host. For example,in constructing a procaryotic expression vector the FIG. 2a secretoryleader is deleted in favor of the bacterial alkaline phosphatase or heatstable enterotoxin II leaders, and for yeast the FIG. 2a leader issubstituted in favor of the yeast invertase, alpha factor or acidphosphatase leaders. This is not to imply, however, that the humansecretory leader is not recognized by hosts other than human cell lines.When the secretory leader is "recognized" by the host, the fusionprotein consisting of lymphotoxin and the leader ordinarily is cleavedat the leader-lymphotoxin peptide bond in the same event that leads tosecretion of the lymphotoxin. Thus, even though a mutant DNA is used totransform the host the resulting product lymphotoxin may be either afused or native lymphotoxin, depending upon the efficacy of the hostcell in processing the fusion.

Another major class of DNA mutants that are not expressed as lymphotoxinvariants are nucleotide substitutions made to enhance expression,primarily by avoiding stem-loop structures in the transcribed mRNA (seecopending U.S. Ser. No. 303,687, now abandoned, incorporated byreference) or to provide codons that are more readily transcribed by theselected host, e.g. the well-known E. coli preference codons for E. coliexpression.

The mutant nucleic acid is made by known methods per se (A. Hui et al.,1984, "The EMBO Journal" 3(3): 623-629; J. Adelman et al., 1983, "DNA"2(3): 183-193; U.K. Patent Application 2,130,219A; G. Winter et al.,1982, "Nature" 299: 756-758; and R. Wallace et al., 1981, "Nucleic AcidsResearch" 9(15): 3647-3656). These methods include M13 phagemutagenesis, synthesis of the mutant lymphotoxin gene as described inExample 1 et seq. or other methods as are or will become known in theart.

Nucleic acid encoding lymphotoxin is any DNA or RNA sequence thatencodes a polypeptide falling within the definition of lymphotoxinherein, whether or not the nucleotide sequences thereof correspond tothe sequences found in nature. In addition, nucleic acid is includedwithin the scope herein that is capable of hybridizing under at leastlow stringency conditions to nucleic acid encoding lymphotoxin, even ifthe hybridizing nucleic acid does not encode a protein otherwise meetingthe definitional requirements for lymphotoxin. An example of the latterwould be a probe that, because of the short length of polypeptide thatit encodes, is incapable of expressing a biologically activelymphotoxin. The nucleic acid encoding lymphotoxin or capable ofhybridizing therewith is prepared by organic synthesis, substantially asshown in Example 1, or obtained from natural sources by probing genomicor cDNA libraries as shown in the Examples.

The lymphotoxin of this invention is made by a process generallyentailing the transformation of a host with a vector bearing the nucleicacid that encodes the desired lymphotoxin. A vector is a replicable DNAconstruct. Vectors are used herein to amplify DNA or to express DNAwhich encodes lymphotoxin. An expression vector is a DNA construct inwhich a DNA sequence encoding lymphotoxin is operably linked to asuitable control sequence capable of effecting the expression oflymphotoxin in a suitable host. Such control sequences include atranscriptional promoter, an optional operator sequence to controltranscription, a sequence encoding suitable mRNA ribosomal bindingsites, and sequences which control termination of transcription andtranslation.

The vector may be a plasmid, a virus.(including phage), or anintegratable DNA fragment (i.e., integratable into the host genome byrecombination). Once transformed into a suitable host, the vectorreplicates and functions independently of the host genome, or may, insome instances, integrate into the genome itself. In the presentspecification, "plasmid" and "vector" are sometimes used interchangeablyas the plasmid is the most commonly used form of vector at present.However, all other forms of vectors which serve an equivalent functionand which are, or become, known in the art are suitable for use herein.

Suitable vectors will contain replicon and control sequences which arederived from species compatible with the intended expression host.Transformed host cells are cells which have been transformed ortransfected with lymphotoxin vectors constructed using recombinant DNAtechniques. Transformed host cells ordinarily express lymphotoxin. Theexpressed lymphotoxin will be deposited intracellularly or secreted intoeither the periplasmic space or the culture supernatant, depending uponthe host cell selected.

DNA regions are operably linked when they are functionally related toeach other. For example, DNA for a presequence or secretory leader isoperably linked to DNA for a polypeptide if it is expressed as apreprotein which participates in the secretion of the polypeptide; apromoter is operably linked to a coding sequence if it controls thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to permittranslation. Generally, operably linked means contiguous and, in thecase of secretory leaders, contiguous and in reading phase.

Suitable host cells are prokaryotes, yeast or higher eukaryotic cells.Prokaryotes include gram negative or gram positive organisms, forexample E. coli or Bacilli. Higher eukaryotic cells include establishedcell lines of mammalian origin as described below. A preferred host cellis the phage resistant E. coli W3110 (ATCC 27,325) strain described inthe Examples, although other prokaryotes such as E. coli B, E. coliX1776 (ATCC 31,537), E. coli 294 (ATCC 31,446), pseudomonas species, orSerratia Marcesans are suitable.

Prokaryotic host-vector systems are preferred for the expression oflymphotoxin. A plethora of suitable microbial vectors are available.Generally, a microbial vector will contain an origin of replicationrecognized by the intended host, a promoter which will function in thehost and a phenotypic selection gene, for example a gene encodingproteins conferring antibiotic resistance or supplying an auxotrophicrequirement. Similar constructs will be manufactured for other hosts. E.coli is typically transformed using pBR322, a plasmid derived from an E.coli species (Bolivar, et al., 1977, "Gene" 2: 95). pBR322 containsgenes for ampicillin and tetracycline resistance and thus provides easymeans for identifying transformed cells.

Expression vectors must contain a promoter which is recognized by thehost organism, but cloning vectors need not. The promoter generally ishomologous to the intended host. Promoters most commonly used inrecombinant DNA construction include the β-lactamase (penicillinase) andlactose promoter systems (Chang et al., 1978, "Nature", 275: 615; andGoeddel et al., 1979, "Nature" 281: 544), a tryptophan (trp) promotersystem (Goeddel et al., 1980, "Nucleic Acids Res." 8: 4057 and EPO App.Publ. No. 36,776) and the tac promoter H. De Boer et al., "Proc. Nat'l.Acad. Sci. U.S.A." 80: 21-25 (1983)!. While these are the most commonlyused, other known microbial promoters are suitable. Details concerningtheir nucleotide sequences have been published, enabling a skilledworker operably to ligate them to DNA encoding lymphotoxin in plasmidvectors (Siebenlist et al., 1980, "Cell" 20: 269) and the DNA encodinglymphotoxin. At the present time the preferred vector is a pBR322derivative containing the E. coli alkaline phosphatase promoter with thetrp Shine-Dalgarno sequence. The promoter and Shine-Dalgarno sequenceare operably linked to the DNA encoding the lymphotoxin, i.e., they arepositioned so as to promote transcription of lymphotoxin mRNA from theDNA.

In addition to prokaryates, eukaryotic microbes such as yeast culturesare transformed with lymphotoxin-encoding vectors. Saccharomycescerevisiae, or common baker's yeast, is the most commonly used amonglower eukaryotic host microorganisms, although a number of other strainsare commonly available. Yeast vectors generally will contain an originof replication from the 2 micron yeast plasmid or an autonomouslyreplicating sequence (ARS), a promoter, DNA encoding lymphotoxin(including in particular human pre lymphotoxin), sequences forpolyadenylation and transcription termination and a selection gene. Asuitable plasmid for lymphotoxin expression in yeast is YRp7,(Stinchcomb et al., 1979, "Nature", 282: 39; Kingsman et al., 1979,"Gene", 7: 141; Tschemper et al., 1980, "Gene", 10: 157). This plasmidalready contains the trp1 gene which provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan, forexample ATCC No. 44076 or PEP4-1 (Jones, 1977, "Genetics", 85: 12). Thepresence of the trp1 lesion in the yeast host cell genome then providesan effective environment for detecting transformation by growth in theabsence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters formetallothionein, 3-phosphoglycerate kinase (Hitzeman et al., 1980, "J.Biol. Chem.", 255: 2073) or other glycolytic enzymes (Hess et al., 1968,"J. Adv. Enzyme Reg.", 7: 149; and Holland et al., 1978, "Biochemistry",17: 4900), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. Suitable vectors and promoters for use in yeast expressionare further described in R. Hitzeman et al., EPO Pubin. No. 73,657.

Other promoters, which have the additional advantage of transcriptioncontrolled by growth conditions, are the promoter regions for alcoholdehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymesassociated with nitrogen metabolism, and the aforementionedmetallothionein and glyceraldehyde-3-phosphate dehydrogenase, as well asenzymes responsible for maltose and galactose utilization. Inconstructing suitable expression plasmids, the termination sequencesassociated with these genes are also ligated into the expression vector3' of the lymphotoxin coding sequences to provide polyadenylation of themRNA and termination.

In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. This, however, is notpreferred because of the excellent results obtained thus far withlymphotoxin expressing microbes. In principal, any higher eukaryoticcell culture is workable, whether from vertebrate or invertebrateculture. However, interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture (tissue culture) has become aroutine procedure in recent years Tissue Culture, Academic Press, Kruseand Patterson, editors (1973)!. Examples of useful host cell lines areVERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and WI38,BHK, COS-7 and MDCK cell lines. Expression vectors for such cellsordinarily include (if necessary) an origin of replication, a promoterlocated upstream from the gene to be expressed, along with a ribosomebinding site, RNA splice site (if intron-containing genomic DNA isused), a polyadenylation site, and a transcriptional terminationsequence.

The transcriptional and translational control sequences in expressionvectors to be used in transforming vertebrate cells are often providedby viral sources. For example, commonly used promoters are derived frompolyoma, Adenovirus 2, and most preferably Simian Virus 40 (SV40). Theearly and late promoters are particularly useful because both areobtained easily from the virus as a fragment which also contains theSV40 viral origin of replication (Fiers et al., 1978, "Nature", 273:113). Smaller or larger SV40 fragments may also be used, provided theapproximately 250 bp sequence extending from the Hind III site towardthe Bgl I site located in the viral origin of replication is included.Further, it is also possible, and often desirable, to utilize the humangenomic promoter, control and/or signal sequences normally associatedwith lymphotoxin, provided such control sequences are compatible withthe host cell systems.

An origin of replication may be provided either by construction of thevector to include an exogenous origin, such as may be derived from SV40or other viral (e.g. Polyoma, Adenovirus, VSV, or BPV) source, or may beprovided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter is oftensufficient. Lymphotoxin is prepared without an amino-terminal methionylby transformation of higher eukaryotic cells with the human prelymphotoxin DNA.

In selecting a preferred host mammalian cell for transfection by vectorswhich comprise DNA sequences encoding both lymphotoxin and dihydrofolatereductase (DHFR), it is appropriate to select the host according to thetype of DHFR protein employed. If wild type DHFR protein is employed, itis preferable to select a host cell which is deficient in DHFR thuspermitting the use of the DHFR coding sequence as a marker forsuccessful transfection in selective medium which lacks hypoxanthine,glycine, and thymidine. An appropriate host cell in this case is theChinese hamster ovary (CHO) cell line deficient in DHFR activity,prepared and propagated as described by Urlaub and Chasin, 1980, "Proc.Natl. Acad. Sci." (USA) 77: 4216.

On the other hand, if DNA encoding DHFR protein with low bindingaffinity for methotrexate (MTX) is used as the controlling sequence, itis not necessary to use DHFR resistant cells. Because the mutant DHFR isresistant to MTX, MTX containing media can be used as a means ofselection provided that the host cells are themselves MTX sensitive.Most eukaryotic cells which are capable of absorbing MTX appear to bemethotrexate sensitive. One such useful cell line is a CHO line, CHO-K1(ATCC No. CCL 61).

Transformed host cells are cells which have been transformed ortransfected with lymphotoxin vectors constructed using recombinant DNAtechniques. Transformed host cells ordinarily express lymphotoxin. Theexpressed lymphotoxin ordinarily is deposited intracellularly.

Lymphotoxin is recovered from recombinant culture in nonsecreting cellsby lysing the cells and removing particulate matter by centrifugation orthe like. Lymphotoxin secreting cells are separated from culturesupernatant by centrifugation. The contaminated lymphotoxin solution isthen purified by the methods referred to above or by immunoaffinity asdescribed in Example 4 below. The lymphotoxin is purified to levelssuitable for pharmacological use and placed into conventional dosageforms, e.g. vials or syringes. Mixtures of lymphotoxin variants areemployed, e.g. a bank of cytotoxic mutant lymphotoxin species.Lymphotoxin optimally is lyophilized for long term storage, or it may beplaced in aqueous solution with stabilizers and excipients, for exampleisotonic saline, and administered to patients as disclosed by B.Aggarwal et al., European Patent Application 100641.

Lymphotoxin compositions are administered to tumor-bearing animals. Theroute of administration is in accord with known methods, e.g.intravenous, intraperitoneal, subcutaneous, intramuscular, intralesionalinfusion or injection of sterile lymphotoxin solutions, or by timedrelease systems described below. Lymphotoxin is administeredintralesionally, i.e., by direct injection into solid tumors. In thecase of disseminated tumors such as leukemia, administration ispreferably intravenous or into the lymphatic system. Tumors of theabdominal organs such as ovarian cancer are advantageously treated byintraperitoneal infusion using peritoneal dialysis hardware andperitoneum-compatible solutions. Ordinarily, however, lymphotoxin isadministered continuously by infusion although bolus injection isacceptable.

Lymphotoxin desirably is administered from an implantable timed-releasearticle. Examples of suitable systems for proteins having the molecularweight of lymphotoxin dimers or trimers include copolymers of L-glutamicacid and gamma ethyl-L-glutamate (U. Sidman et al., 1983, "Biopolymers"22 (1): 547-556), poly (2-hydroxyethyl-methacrylate) (R. Langer et al.,1981, "J. Biomed. Mater. Res." 15: 167-277 and R. Langer, 1982, "Chem.Tech." 12: 98-105) or ethylene vinyl acetate (R Langer et al.,Id.).Lymphotoxin-containing articles are implanted at surgical sites fromwhich tumors have been excised. Alternatively, lymphotoxin isencapsulated in semipermeable microcapsules or liposomes for injectioninto the tumor. This mode of administration is particularly useful forsurgically inexcisable tumors, e.g. brain tumors.

The amount of lymphotoxin that is administered will depend, for example,upon the route of administration, the tumor in question and thecondition of the patient. It will be necessary for the therapist totiter the dosage and modify the route of administration as required toobtain optimal cytotoxic activity towards the target tumor, as can bedetermined for example by biopsy of the tumor or diagnostic assays forputative cancer markers such as carcinoembryonic antigen, in view of anyrecombinant toxicity encountered at elevated dosage. Ordinarily,recombinant lymphotoxin dosages in mice at about from 50 to 200 μg/kgbody weight/day by intravenous administration have been found to besubstantially nontoxic and efficacious in vivo. Obviously, the dosageregimen will vary for different animals.

A method is provided herein for obtaining lymphotoxin-neutralizingantibody. Neutralizing antibody is defined as antibody that is capableof immunologically binding lymphotoxin as defined herein in such a wayas to substantially reduce its activity in cytostatic or cytolyticlymphotoxin activity assays such as the murine L929 assay describedbelow. The fact that the antibody is capable of neutralizing lymphotoxinactivity does not mean that the antibody must bind directly to thelymphotoxin active or receptor binding site. The antibody may stillsubstantially neutralize lymphotoxin activity if it sterically binds toa region which adjacent to the critical site, i.e., adjacent in thesense of conformationally adjacent and not necessarily adjacent from thepoint of view of amino acid sequence.

In attempting to prepare a neutralizing monoclonal antibody againstlymphotoxin, it proved difficult to immunize mice in a fashion such thatlymphotoxin neutralizing antibody is generated or raised in the animals.Neither immunization with lymphoblastoid lymphotoxin nor glutaraldehydecross-linked lymphotoxin resulted in any detectable neutralizingantibody in the serum of immunized mice, even though the mice did raisenon-neutralizing anti-lymphotoxin antibody detectable by enzymeimmunoassay. However, immunization with a lymphotoxin-alum (aluminumhydroxide or alumina, Al₂ O₃.3H₂ O) adsorption complex will raiseneutralizing antibody even in animals which had failed to generate theactivity prior to immunization with the alum complex. Preparation ofalum and its use in the production of antiserum are disclosed in C.Williams, et al.,eds., 1967, Methods in Immunology and ImmunochemistryI, pp 197-229.

Fusions of spleen cells from animals producing neutralizing antibodywith murine myeloma cells are made. On the average, about 50 to 100clones will have to be screened to identify one which synthesizesneutralizing antibody. The process for screening the clones for thedesired activity is routine and well within the skill of the ordinaryartisan, and can be reproduced with minimal experimental effort.

The serum, plasma or IgG fractions from the immunized animal, as well asimmunoglobulins secreted by hybridomas generated from the spleen orlymph cell of immunized animals, are all satisfactory for use herein. Ina preferred embodiment the neutralizing antibody is obtained essentiallyfree of other anti-lymphotoxin antibody in hybridoma culture.

The neutralizing antibody is immobilized by adsorption to surfaces,e.g., thermoplastics such as polystyrene, or covalently bound tomatrices such as cyanogen bromide-activated Sepharose. It then is usedin immunoassays or in immunoaffinity purification. Since the antibody isa neutralizing antibody it is most likely only to adsorb or detectbiologically active lymphotoxin or fragments thereof. The antibody isparticularly useful in immunoradiometric ("sandwich") immunoassays inconcert with a non-neutralizing anti-lymphotoxin monoclonal antibody ora polyclonal antiserum which contains non-neutralizing anti-lymphotoxin.The immunoassay is conducted using either the neutralizing ornon-neutralizing antibody as the labelled component, which labelling iseffective with a detectable substance such as a fluorescent,chemiluminescent or radioisotopic label in accord with methods known inthe art. For competitive-type lymphotoxin immunoassays, lymphotoxin islabelled in the same fashion. Chloramine-T radioiodination is suitablefor both lymphotoxin and lymphotoxin antibody tracer preparation, or themethod described in J. Klostergaard et al., "Mol. Immun." 18: 455 (1980)is used.

In order to simplify the Examples certain frequently occurring methodswill be referenced by shorthand phrases.

Plasmids are designated by a low case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein arecommercially available, are publically available on an unrestrictedbasis, or can be constructed from such available plasmids in accord withpublished procedures. In addition, other equivalent plasmids are knownin the art and will be apparent to the ordinary artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with anenzyme that acts only at certain locations in the DNA. Such enzymes arecalled restriction enzymes, and the sites for which each is specific iscalled a restriction site. "Partial" digestion refers to incompletedigestion by a restriction enzyme, i.e., conditions are chosen thatresult in cleavage of some but not all of the sites for a givenrestriction endonuclease in a DNA substrate. The various restrictionenzymes used herein are commercially available and their reactionconditions, cofactors and other requirements as established by theenzyme suppliers were used. Restriction enzymes commonly are designatedby abbreviations composed of a capital letter followed by other lettersand then, generally, a number representing the microorganism from whicheach restriction enzyme originally was obtained. In general, about 1 μgof plasmid or DNA fragment is used with about 1 unit of enzyme in about20 μl of buffer solution. Appropriate buffers and substrate amounts forparticular restriction enzymes are specified by the manufacturer.Incubation times of about 1 hour at 37° C. are ordinarily used, but mayvary in accordance with the supplier's instructions. After incubation,protein is removed by extraction with phenol and chloroform, and thedigested nucleic acid is recovered from the aqueous fraction byprecipitation with ethanol. Digestion with a restriction enzymeinfrequently is followed with bacterial alkaline phosphatase hydrolysisof the terminal 5' phosphates to prevent the two restriction cleavedends of a DNA fragment from "circularizing" or forming a closed loopthat would impede insertion of another DNA fragment at the restrictionsite. Unless otherwise stated, digestion of plasmids is not followed by5' terminal dephosphorylation. Procedures and reagents fordephosphorylation are conventional (T. Maniatis et al., 1982, MolecularCloning pp. 133-134).

"Recovery" or "isolation" of a given fragment of DNA from a restrictiondigest means separation of the digest on polyacrylamide gelelectrophoresis, identification of the fragment of interest bycomparison of its mobility versus that of marker DNA fragments of knownmolecular weight, removal of the gel section containing the desiredfragment, and separation of the gel from DNA. This procedure is knowngenerally. For example, see R. Lawn et al., 1981, "Nucleic Acids Res."9:6103-6114, and D. Goeddel et al., 1980, "Nucleic Acids Res." 8:4057.

"Southern Analysis" is a method by which the presence of DNA sequencesin a digest or DNA-containing composition is confirmed by hybridizationto a known, labelled oligonucleotide or DNA fragment. For the purposesherein, unless otherwise provided, Southern analysis shall meanseparation of digests on 1 percent agarose, denaturation and transfer tonitrocellulose by the method of E. Southern, 1975, "J. Mol. Biol."98:503-517, and hybridization as described by T. Maniatis et al., 1978,"Cell" 15:687-701.

"Transformation" means introducing DNA into an organism so that the DNAis replicable, either as an extrachromosomal element or chromosomalintegrant. Unless otherwise provided, the method used herein fortransformation of E. coli is the CaCl₂ method of Mandel et al., 1970,"J. Mol. Biol." 53:154.

"Ligation" refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (T. Maniatis et al., Id., p.146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units of T4 DNA ligase ("ligase")per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

"Preparation" of DNA from transformants means isolating plasmid DNA frommicrobial culture. Unless otherwise provided, the alkaline/SDS method ofNaniatis et al., Id. p. 90., may be used.

"Oligonucleotides" are short length single or double strandedpolydeoxynucleotides which are chemically synthesized by the methodincorporated by reference into Example 1, and then purified onpolyacrylamide gels.

All literature citations are expressly incorporated by reference.

EXAMPLE 1 Purification and Sequencing of Lymphotoxin

The human lymphoblastoid cell line RPMI-1788 (ATCC No. CCL-156) alasgrown in 15L spinner flasks to a cell density of 4×10⁵ cells per mlusing a serum free culture medium (RPMI-1640). Lymphotoxin was induced10-20 fold (to 500-1000 lymphotoxin units/ml, determined as describedbelow) over basal levels by the inclusion of 20 ng/ml of phorbolnyristate acetate in the serum free RPMI-1640 medium. After 65 h ofculture, the cells were harvested by filtration, and the lymphotoxinactivity in the filtrate was absorbed to controlled pore glass beads(Electronucleonics) in a column (5 cm×20 cm), equilibrated with 5 mMphosphate buffer (pH 7.4) and eluted with 50 percent ethylene glycol in5 mM phosphate buffer (pH 7.4). 0.1 mM phenylmethyl sulfonyl fluoride(PMSF), a protease inhibitor, and 1 mM sodium azide, for inhibition ofmicrobial growth, were included in all buffers throughout thepurification. The eluate from glass beads contained 84,000 units oflymphotoxin/mg protein. This was followed by DEAE cellulosechromatography, Lentil Lectin Sepharose chromatography, and preparativenative PAGE as described in B. Aggarwal, et al., 1984, "J. Biol. Chem."259 (1): 686-691. Homogeneity of the protein responsible for cytotoxicactivity was determined by SDS-PAGE, reverse-phase HPLC on a LichrosorbRP-18 column and by amino terminal sequencing.

This lymphotoxin preparation contained greater than 95 percent by weightof the leucyl amino-terminal lymphotoxin having an approximate molecularweight of 25,000 on SDS-PAGE. The theoretical molecular weight of theprotein component of the N-terminal leucyl species is 18,664 daltons;the remaining approximately 6,500 daltons was attributed to a glycosylside chain at Asn+62, and perhaps other O-linked sugar residues. Thetissue culture supernatant contained putative multimers of this species(60,000 Da by TSK-HPLC or 64,000 Da by Sephadex G-100 chromatography).

The remaining 5 percent of the lymphotoxin mixture was the N-terminalhistidyl species having a molecular weight of about 20,000. Both speciesexhibit substantially the same cytolytic activity, at least within thelimits of the variation inherent in the murine fibroblast cell lysisassay described below.

Tryptic digestion of the intact lymphotoxin molecules yielded only a fewfragments. Histidyl amino-terminal lymphotoxin was digested into twofragments between amino acid positions 89 and 90, while the leucylamino-terminal tryptic digestion yielded four fragments cleaved betweenpositions 15 and 16, 19 and 20, and 89 and 90.

Micro-sequencing by the Edman degradation technique yielded sequenceinformation on the intact molecule and also on the fragments produced bytryptic cleavage.

Further sequence information was provided by fragments of lymphotoxinproduced by carboxypeptidase P and chymotrypsin digestion, acetic aciddigestion and cyanogen bromide cleavage. Nearly the entire sequence ofthe human lymphotoxin was determined by this method. 156 contiguousresidues were determined from the amino terminus. It was clear from thissequencing information that the difference between the two lymphotoxinspecies was the presence of 23 amino-terminal residues in the leucylamino-terminal species which were not found in the histidylamino-terminal species. The carboxyl terminal sequence beyond the firstthree residues proved to be difficult to determine because of certainpeptide bonds present in this region and the hydrophobic nature of theresidues.

A synthetic gene was designed which would code for the protein sequenceto the extent determined by micro-sequencing. The gene designincorporated a general E. coli codon bias, that is, rarely used E. colicodons were not used in the sequence. Human preference codons weresubstituted where no E. coli codon bias was apparent. This bias waschosen to aid in expression in E. coli, and also so that the syntheticgene would be useful as a probe to identify the natural DNA sequencefrom human cDNA or genomic libraries. The unique restriction sites XbaI,BamHI, HindIII, and BglII were designed into the sequence to aid in theconstruction of the fragments and to allow for future manipulation ofthe gene.

The 58 original oligomers designed for the synthetic lymphotoxin genewere synthesized by the solid phase phosphite method of M. Matteucci etal., 1981, "J. Amer. Chem. Soc." 103: 3185-3190 and S. Beaucage et al.,1981, "Tet. Letters" 22: 1859-1862. The size of these oligomers rangedfrom 16 bases to 20 bases and is shown in FIG. 1a. Overlaps betweenoligomers were 6 bases in length and designed to be unique. The entiregene was assembled as shown in FIG. 1b.

The gene was constructed in three separate pieces. The first, Segment A,was 117 base pairs in length and represented the 5' coding region forthe amino terminal end of the leucyl amino-terminal species. Segment Brepresented the DNA encoding the middle of the lymphotoxin molecule andwas 145 base pairs in length. Segment C, at 217 base pairs in length,was believed to encode all but 16 amino acid residues at the lymphotoxincarboxy terminus. The oligomers required to synthesize each of thesegments were purified by electrophoresis and then pooled. Therelatively small size of each oligomer (that is, 16 to 20 bases) waschosen to reduce errors in synthesis.

Each group of oligomers was phosphorylated in a reaction containing 20mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 20 mM dithiothreitol, 0.5 mM ATP, and15 units of T4 polynucleotide kinase in a volume of 50 μl; approximately50 pmol of each oligomer was contained in the reaction. After 30 minutesat 37° C., the reaction was heated to 65° C. to destroy kinase activity,and then allowed to slowly cool to 20° C. over the period of one hour.The phosphorylated oligomers were then ligated by the addition of 10units of T4 DNA ligase and the reaction was allowed to proceed for 2hours at 20° C. The DNA ligase was heat inactivated and then the ligatedoligomers were digested for 3 hours at 37° C. with restrictionendonucleases which recognized the designed terminal sites (e.g., XbaIand BamHI for segment A). Fragments for each segment were isolated byelectrophoresis on a 7 percent polyacrylamide gel. Fragments of thecorrect mobility were identified for each segment by ethidium bromidestaining and electroeluted from the gel. pFIFtrp69 (D. Goeddel et al.,1980, "Nature" 287: 411-416 or Crea et al., European Patent Application0048970) was digested with XbaI and BamHI and the large vector fragmentisolated by 6 percent polyacrylamide gel electrophoresis. About 50 ng ofsegment A was ligated to the pFIFtrp69 fragment. Similarly, segment Bwas ligated into BamHI and HindIII digested pBR322, and segment C wasligated into HindIII and BglII digested pLeIFA-125-1 (D. Goeddel et al.,1980, "Nuc. Acids Res." 8: 4057-4073). The ligation reaction mixtureswere transformed into E. coli ATCC 31446 and the resulting recombinantplasmids were characterized by restriction endonuclease analysis and DNAsequencing by the Maxam and Gilbert chemical degradation method. Five ofsix segment A clones contained the designed sequence. Four segment B andfour segment C plasmids were isolated, and all of these inserts had thecorrect sequences. Each segment was isolated by digestion withrestriction endonucleases which recognized the terminal sites and thenligated into the plasmid vector pFIFtrp69 digested with XbaI and BglII.The resulting recombinant plasmid, pLTXB1, was characterized bysequencing the inserted XbaI-BglII fragment, which contained thesequence presented in FIG. 1a.

To determine if the synthetic gene would indeed produce biologicallyactive lymphotoxin, the E. coli pLTXB1 transformants were grown inminimal media under conditions to de-repress the trp promoter and allowexpression of the synthetic lymphotoxin gene. Cultures were grown to anoptical density of 1.0 at 550 nanometers and harvested bycentrifugation. The cell pellet was suspended in one-tenth volume, andthen lysed by sonication.

Lymphotoxin activity was determined by the modified cell-lytic assay ofB. Spofford, 1974, "J. Immunol." 112: 2111. Briefly, mouse L-929fibroblast cells were grown in microtiter plates in the presence ofactinomycin D. After 12-18 hours, 0.125 ml of serially diluted sample tobe assayed for lymphotoxin is added to each well. After 18 hours, theplates were washed and the lysis of the cells induced by lymphotoxin wasdetected as adhering to the plates by staining the plates with a 1percent solution of crystal violet in methanol:water (1:4 v/v). Theintensity of stain was observed both visually as well asspectrophotometrically at absorbance of 450 nm and 570 nm transmissionusing a Dynatech spectrophotometer. The cells plated in a microtiterwell with culture medium alone were set at 0 percent lysis whereas thosewith 3M guanidine hydrochloride solution provided an end point for 100percent lysis. One unit of lymphotoxin is defined as the amount requiredfor 50 percent cell lysis out of 12,000 cells plated in each well. Notethat other assays of cytotoxic activity also may be used. For examplesee B. Aggarwal et al., in "Thymic Hormones and Lymphokines", 1983, ed.A. Goldstein, Spring Symposium on Health Sciences, George WashingtonUniv. Medical Center (the A549 cell line referred to in this material isavailable from the ATCC as CCL185). Culture lysates showed undetectablecytolytic activity in the murine cell assay described above. Controllysates from gamma interferon expressing cultures did contain gammainterferon activity. This result suggested that the synthetic gene didnot encode an active lymphotoxin. There were several possibleexplanations for this. For example: (1) the E. coli degraded thelymphotoxin, (2) the lymphotoxin gene was not transcribed in E. coli,(3) the lymphotoxin message was not translated in E. coli, (4) theprotein did not have the proper sequence due to a protein sequencingerror, or (5) the 16 residue carboxy terminal sequence or a portionthereof was actually necessary for activity or for proper configurationof the lymphotoxin molecule.

EXAMPLE 2 Procedure for Obtaining cDNA Encoding Lymphotoxin

RNA was isolated from a culture of a non-adherent cell fraction of humanperipheral blood lymphocytes 48 hours after induction with phorbolmyristate acetate (10 ng/ml), staphylococcal enterotoxin B (1 μg/ml) andthymosin α-1 (S. Berger et al., 1979, "Biochemistry" 18: 5143-5149).This culture was producing 400 units of lymphotoxin activity/ml ofsupernatant. The mRNA was concentrated by adsorption to immobilizedoligo dT, eluted and cDNA prepared by reverse transcription (P. Gray etal., 1982, "Nature" 295: 503-508). Reverse transcriptase was used tomake a cDNA copy of the messenger RNA by standard methods, a secondstrand was prepared (also by standard methods) by Klenow treatment, andthe cDNA was treated with S-1 nuclease to remove the hairpin loop. Inorder to insert this cDNA into a vector the ends were ligated to anadaptor or linker so as to create 5' and 3' restriction enzyme sites or,preferably, cohesive terminii for a predetermined restriction enzymesite. The oligonucleotide 5' HO-AATTCATGCGTTCTTACAGGTACGCAAGAATGTC-P 5'was used for this purpose. The oligonucleotide was ligated to the cDNAand the cDNA reisolated by polyacrylamide gel electrophoresis. λgt10, apublicly available phage (or its substantial equivalent, λgt11, which isavailable from the ATCC), was digested with EcoRI and the linearfragment recovered (M. Wickens et al., 1978, "J.Biol. Chem." 253:2483-2495). The Tinkered reverse transcript and the λgt10 digest wereligated and the ligation mixture used to transfect E. coli C-600 orother known host susceptible to λ phage infection. Approximately 10,000recombinant phage were plated on a 15 cm plate and screened by alow-stringency plaque hybridization method (T. Maniatis et al., 1978,"Cell" 15: 687-701 and P. Gray et al., "PNAS" 80: 5842-5846) using a ³²P-labelled probe prepared from Segment A of FIG. 1a by the method of J.Taylor et al., 1976, "Biochem. Biophys. Acta" 442: 324-330 in which calfthymus DNA primers were used (PL Biochemicals). Duplicate nitrocellulosefilters were hybridized by the low stringency method with 5×10⁷ cpm ofthe probe in 20 percent formamide. The filters were washed twice in 0.3Msodium chloride, 0.03M sodium citrate, and 0.1 percent sodium dodecylsulfonate (SDS) at 37° C.

Two phages hybridized with the probe and were plaque purified. Thepurified phage hybridized with both the Segment A probe and a probeprepared from Segment B. The cDNA inserts of the two hybridizing phages,λLT1 and λLT2, were subcloned into M13mp8 and sequenced by the dideoxychain termination method (A. Smith, 1980, "Methods in Enzymology" 65:560-580). The insert in λLT2 was only about 600 bp and did not containthe entire 3' coding region for lymphotoxin. The insert in λLT1contained the entire coding region for leucyl amino-terminal lymphotoxinplus a 650 bp 3' untranslated region (containing a consensuspolyadenylation signal) and codons for 18 amino acids amino terminal tothe leucyl terminus. Since this did not constitute the entirelymphotoxin coding region an additional ³² P-labelled probe was preparedfrom the cDNA insert of λLT1 and used to screen an additional 25,000recombinant λgt10 phage at high stringency (see T. Huynh et al., 1984,in Practical Approaches in Biochemistry IRL Press, Oxford). Twelveadditional hybridizing phages were isolated and the sequence of thelongest insert, from λLT11, is presented in FIG. 2a. The longest openreading frame was translated starting at the first observed ATG. Numbersabove each line refer to amino acid position and numbers below each linerefer to nucleotide position. The leucyl residue labelled "1" representsthe first residue sequenced of leucyl amino-terminal lymphotoxin (FIG.1a) and is presumably the first amino terminal residue of the maturespecies of lymphotoxin. The first 34 residues represent a signalsequence. Residues 156-171 had not been determinable by proteinsequencing of lymphotoxin, but instead were imputed from the nucleotidesequence.

EXAMPLE 3 Construction of a Hybrid Synthetic Gene/Natural cDNAExpression Vector for Leucyl Amino-Terminal Lymphotoxin

This construction is shown in FIG. 2b. pLTXB1 (containing the inactivesynthetic gene) was partially digested with EcoRI and PstI, and a 685 bpfragment containing DNA encoding 125 N-terminal residues of lymphotoxinwas recovered. A partial PstI digest was performed because of thepresence of an additional PstI site at residue 10 (FIG. 1a). A 301 bpfragment containing DNA encoding the C-terminal 51 amino acids oflymphotoxin was isolated by digesting the subcloned cDNA of λLT1 withEcoRI and PstI (these sites are shown above in FIG. 2a at nucleotidepositions 554 and 855). These fragments were isolated by electrophoresison 5 percent polyacrylamide and electroelution. The fragments wereligated into pBR322 which had been digested with EcoRI anddephosphorylated with bacterial alkaline phosphatase to reducebackground transformants. The resulting expression plasmid, pLTtrp1, wascharacterized as to proper orientation and sequence by restrictionendonuclease digestion and DNA sequencing. Leucyl amino-terminallymphotoxin was expressed by transforming E. coli 31446 with pLTtrp1 andculturing the transformants in medium containing tetracycline at 37° C.for 4-6 hours until an OD. of 1.0 was reached. The cell lysatescontained cytotoxic activity. The leucyl amino terminus of the expressedlymphotoxin species was found to be substituted with a blocked methionylresidue. It is believed that the product of this synthesis is the formylmethionyl rather than methionyl species.

EXAMPLE 4 Immunoaffinity Purification of Lymphotoxin

A murine monoclonal cell line secreting anti-lymphotoxin (Example 8) wasgrown in mice and purified from ascites fluid by ion exchangechromatography. The anion exchange eluate was coupled to cyanogenbromide activated Sepharose at a concentration of 2 mg/ml resin. A 20 mlcolumn was equilibrated consecutively with TBS (containing 0.05MTris-HCl, pH 7.0, 0.15M sodium chloride, and 2 mM EDTA); then withelution buffer (containing 0.1M acetic acid, pH 4.5, 150 mM sodiumchloride); and finally with TBS. A 40 percent saturated ammonium sulfateprecipitate of pLTtrp1-transformed E. coli sonicated lysate (previouslyclarified by centrifugation) was suspended in 0.1M Tris-HCl, pH 7.4, and5 mM EDTA and loaded onto the column at a rate of one column volume perhour. Following extensive washing with TBS containing 0.05 percentTween-20, specifically bound material was eluted with the elutionbuffer, the pH immediately adjusted to 7.8 with 0.1 volume 1M Tris-HCl,pH 8.5, and stored at 4° C. The specific activity of this purifiedlymphotoxin was 2-10×10⁷ units/mg, as measured in the above murine L-929assay.

The eluate contained most of the activity loaded onto the column. Themajority of the total eluate protein migrated as a single band underboth reducing and nonreducing conditions in SDS-polyacrylamide gelelectrophoresis. The mobility of this band corresponds to approximately18,000 NW, which is consistent with the predicted value of 18,664 MW furunglycosylated leucyl-amino terminal lymphotoxin based on the deducedamino acid sequence. To further characterize its biological activities,the purified recombinant lymphotoxin was tested for cytolytic activityin vitro and antitumor activity in vivo.

EXAMPLE 5 In Vivo Biological Activity of Recombinant Lymphotoxin

Recombinant and lymphoblastoid lymphotoxin were tested in an in vivotumor necrosis assay. MethA(a) sarcomas were grown for 7-10 days insusceptible mice BALB/C×C57B1/6fl or CB6fl!, and the tumors thendirectly injected with Example 4 lymphotoxin, lymphoblastoid lymphotoxin(prepared and purified as described above) or control samples. After20-24 hours, the mice were sacrificed, the tumors removed andhistologically scored for the extent of necrosis. As shown in Table 1,both recombinant and lymphoblastoid lymphotoxin caused significantnecrosis of MethA(a) sarcoma in vivo. Control samples did not inducenecrosis of the MethA(a) sarcomas.

                  TABLE 1                                                         ______________________________________                                        NECROSIS OF MethA(a) SARCOMA IN VIVO BY RECOMBINANT                           AND NATURAL LYMPHOTOXIN                                                                        Number of Mice                                                                Sarcoma Necrosis Score                                       Treatment          +++    ++      +   -                                       ______________________________________                                        Buffer 1 control   --     --      --  3                                       Lymphoblastoid Lymphotoxin,                                                                      4      --      --  --                                      25,000 units                                                                  Lymphoblastoid Lymphotoxin,                                                                      4      --      --  --                                      10,000 units                                                                  Recombinant Lymphotoxin,                                                                         14     2       2   --                                      200,000 units                                                                 Recombinant Lymphotoxin,                                                                         3      --      --  1                                       25,000 units                                                                  Recombinant Lymphotoxin,                                                                         3      --      1   --                                      10,000 units                                                                  Buffer 2 Control   --     --      --  9                                       ______________________________________                                    

Lymphoblastoid lymphotoxin was injected dissolved in buffer 1 (0.01MTris-HCl, 0.05M (NH₄)₂ HCO₃, pH 8.0) and recombinant lymphotoxin wasinjected dissolved in Buffer 2 (0.15M NaCl, 0.1M sodium acetate and 0.1MTris-HCl, pH 7.8).

The absence of carbohydrate on recombinant lymphotoxin does not appearto affect biological activity, since the specific activity oflymphotoxin produced by recombinant culture (2-10×10⁷ units/mg) isapproximately the same as that reported for lymphoblastoid lymphotoxin(4×10⁷ units/mg).

The recombinant lymphotoxin activity also exhibited thermolabilitysimilar to natural lymphotoxin, i.e., inactivation in aqueous solutionafter heating for 1 hour at 80° C.

EXAMPLE 6 Construction of an Expression Vector for Methionyl HistidylAmino-Terminal Lymphotoxin

Construction of a plasmid which directs the expression in E. coli ofmethionyl histidyl amino-terminal lymphotoxin is outlined in FIG. 3. Asynthetic oligonucleotide was Inserted into the expression plasmid so asto encode an initiator methionine codon adjadent to the histidyl codonof histidyl amino terminal lymphotoxin (residue 24 of FIG. 2a). This wasperformed by isolating a 4630 bp vector fragment from pLTtrp1 by XbaIand ClaI digestion, preparative 1 percent agarose gel electrophoresis,and electroelution. A 570 bp BamHI-ClaI fragment containing most of thelymphotoxin coding sequence was also isolated from pLTtrp1 in the samefashion. Two synthetic oligonucleotides were synthesized by methodsdiscussed previously and mixed with oligonucleotides 6, 7, 52 and 53 ofFIG. 1a. Approximately 50 pmol of each oligonucleotide was treated withpolynucleotide kinase as described in Example 1. The oligonucleotideswere annealed and then ligated with a mixture of the 570 bp BamHI-ClaIfragment and the 4630 bp XbaI-ClaI vector fragment. The ligation mixturewas transformed into E. coli ATCC 31446 and recombinants were selectedon the basis of resistance to tetracycline. Plasmid p20KLT was recoveredfrom one of the transformants. Plasmid p20KLT was characterized byrestriction enzyme and DNA sequence analysis.

EXAMPLE 7 Preparation of Cytotoxic Lymphotoxin Fusion Variant

A plasmid containing DNA encoding a fusion of lymphotoxin with abacterial protein was constructed by cloning a sequence coding for abacterial signal sequence adjacent to the structural gene forlymphotoxin. The sequence of the gene for the heat-stable Enterotoxin II(STII) of E. coli has been characterized (R. N. Picken et al., 1983,"Infection and Immunity" 42: 269-275) and encodes a 23 amino acid signalsequence which directs the secretion of the STII into the periplasmicspace of E. coli.

The plasmid pWM501 (Picken et al., 1983, "Infection and Immunity" 42 1!:269-275) contains the heat-stable enterotoxin (STII) gene. A portion ofthe DNA which encodes the STII gene was recovered from pWM501 using thefollowing steps. pWM501 was digested with RsaI and the 550 bp DNAfragment was isolated. This gene fragment was ligated to the phageM13mp8 (J. Messing et al. in the Third Cleveland Symposium onMacromolecules: Recombinant DNA, Ed. A. Walton, Elsevier, Amsterdam1981! pp 143-153) that had been previously digested with SmaI. Theligated DNA was used to transform E. coli JM101, a commerciallyavailable strain for use with the M13 phage. Clear plaques wererecovered. The double stranded M13mp8 STII Rsa derivative was isolatedfrom an E. coli JM101 infected with this phage using standard procedures(J. Messing et al. op cit). By the use of the M13mp8 subcloningprocedure just described the approximately 550 base pair fragmentcontaining the STII leader gene is now bounded by a series of differentrestriction endonuclease sites provided by the phage. The M13mp8 STIIRsa derivative then was digested with EcoRI and Pst I and a DNA fragmentslightly larger than the 550 bp DNA fragment was isolated.

The EcoRI-PstI fragment was subcloned into pBR322. This was accomplishedby digesting pBR322 with EcoRI and PstI and isolating the vector. Theisolated vector was ligated to the EcoRI-PstI DNA fragment. This DNAmixture was used to transform E. coli ATCC 31446 and tetracyclineresistant colonies selected. A plasmid was isolated from a resistant E.coli colony and designated pSTII-partial.

pSTII-partial was digested with MnlI and BamHI and a 180 bp fragmentcontaining the STII Shine-Dalgarno sequence, the STII signal sequence,and the first 30 codons of the mature STII gene was isolated. The 180 bpDNA fragment was ligated to a plasmid containing the trp promoter. Onesuch plasmid, pHGH207-1, has been described previously (H. de Boer etal., 1982, in: Promoters: Structure and Function, Eds. R. Rodreguez etal. Chamberlin, Praeger Pub., New York, N.Y., pp 462-481). A derivativeof this plasmid, pHGH207-1*, wherein the EcoRI site 5' to the trppromoter had been converted to EcoRI* by filling in with DNA polymeraseI (DNA pol I) and joining the blunt ends by ligation (S. Cabilly et al.,1984, "Proc. Natl. Acad. Sci. USA" 81: 3273-3277) was used in thisexample. The trp promoter-containing plasmid was digested with XbaI andtreated with DNA pol I and all four dNTPs to fill in the protrudingsequence. The DNA preparation was then digested with BamHI and thevector-containing fragment isolated. This vector fragment then wasligated to the 180 bp STII signal-containing DNA fragment isolatedabove. The ligation mixture was used to transform E. coli ATCC 31446 toampicillin resistance. A plasmid designated STII-leader was isolatedfrom an ampicillin resistant colony.

An M13 phage containing STII encoding sequences was first constructed byligating the 180 bp XbaI-BamHI fragment of pSTII-leader into XbaI andBamHI digested M13mp10. The resulting phage DNA, pSTII-shuttle, wascharacterized by restriction endonuclease analysis and nucleotidesequencing. LT encoding sequences were then introduced into this vectorby ligating the HpaI-EcoRI 700 bp fragment of pLTtrp1 into SmaI-EcoRIdigested pSTII-shuttle replicative form (RF, double stranded) DNA; SmaIand HpaI sites are both blunt ended and ligated together (resulting inthe loss of both sites). The resulting phage DNA, M13-STII-LT, wascharacterized and then utilized for mutagenesis as follows: the primer5' p CAAATGCCTATGCACTGCCAGGCGTAGG was kinased and mixed with thetemplate (M13-STII-LT) in the presence of ligase buffer and XbaI-EcoRIdigested M13mp10 RF DNA (to promote priming of DNA, as reported by J. P.Adelman et al., 1983, "DNA" 2: 183-193); the mixture was heated to 95°C. and then allowed to anneal at room temperature for 30 minutes andthen placed on ice for 30 minutes. All four deoxynucleotidetriphosphates were then added along with ATP, T4 DNA Ligase, and thelarge fragment (Klenow) of E. coli DNA polymerase I. The mixture wasincubated 1 hour at 14° C. and then used to transfect competent E. coliJM101, a commercially available strain, or any other M13 phage host.Correctly mutagenized phage were identified by hybridization screeningutilizing the ³² P-radiolabeled-primer as a probe. The resulting phageST-LT-mut was characterized by DNA sequence analysis. Replicative formDNA was prepared from this phage and used for isolation of a 761 bpXbaI-EcoRI fragment containing DNA for the STII signal sequence adjacentto the coding sequence of Leucyl-amino terminal lymphotoxin. This DNAwas ligated with XbaI-BamHI digested p20KLT (the large 4285 bp vectorfragment) and the 375 bp EcoRI-BamHI fragment of pBR322. The resultingplasmid, pST18LT, was characterized by restriction mapping and DNAsequencing. A similar construction was prepared that encoded a fusion ofthe STII signal amino-terminal to the histidine residue of histidylamino-terminal lymphotoxin. The resulting plasmids were transformed intoE. coli ATCC 31446. Plasmids pSTLT18 and pSTLT16 were recovered. Theywere confirmed to encode the STII fusion by restriction enzyme analysisand dideoxy sequencing. E. coli transformed with plasmids pSTLT18 orpSTLT16 synthesize STII signal sequence fusions with leucylamino-terminal and histidyl amino-terminal lymphotoxin as determined tobe consistent with the calculated molecular weights by gelelectrophoresis. The E. coli lysates containing these fusion proteinsexhibited cytotoxic activity.

EXAMPLE 8 Method for Making Monoclonal Murine Antibody Capable ofNeutralizing Lymphotoxin

Purified lymphoblastoid lymphotoxin obtained in Example 1 was dialyzedagainst phosphate buffered saline (PBS). 200 μg of lymphotoxin/ml werecontained in the dialysate. Glutaraldehyde was added to the dialysate toa concentration of 70 mM glutaraldehyde, the mixture incubated for 2hours at room temperature, more glutaraldehyde added to bring its totaladded concentration up to 140 mM, incubation continued for another 6hours and the mixture then dialyzed against PBS. 50 μg of theglutaraldehyde cross-linked lymphotoxin (hereafter, "polylymphotoxin")and 0.5 ml of Freund's complete adjuvant was injected subcutaneouslyinto mice (strain BALB/c). After one week, the mouse was boosterimmunized with 50 μg of polylymphotoxin and 0.5 ml of Freund'sincomplete adjuvant, half intramuscularly and half into the peritonealcavity. Serum was harvested after 7 days and assayed foranti-lymphotoxin activity by an ELISA assay.

The ELISA assay was conducted as follows: A buffered solution ofpurified lymphotoxin was placed in microtiter wells and permitted tocoat the wells with about 100 ng of lymphotoxin each. The unadsorbedlymphotoxin solution was aspirated from the wells. 50 μl ofappropriately diluted test sample was combined with 100 μl PBScontaining 5 mg/ml bovine serum albumin (PBS-BSA buffer) and added toeach well, incubated for 2 hours at room temperature, washed with PBScontaining 0.05 percent Tween 20, 100 μl of horse radishperoxidase-labelled goat anti-mouse IgG in PBS-BSA buffer added to eachwell and incubated for 1 hour. Each well was washed with PBS containing0.05 percent Tween 20 and then citrate phosphate buffer, pH5, containing0.1 mg o-phenylene diamine/ml (substrate solution) and aqueous 30percent H₂ O₂ (at a proportion of 4 μl of 30 percent v/v H₂ O₂ per 10 mlof substrate solution) was added to each well. The wells were incubatedfor 30 min., the reaction stopped with 50 μl 2.5M sulfuric acid andadsorbance measured at 492 nm. Wells which showed adsorbance greaterthan 1 O.D. were considered anti-lymphotoxin positive.

Test samples also were assayed for the ability to neutralize thecytolytic activity of lymphotoxin in the murine L929 assay. Serumharvested from immunized animals or hybridoma supernatants were dilutedas required into RPMI-1640 medium containing 10 percent fetal bovineserum and about 100 lymphotoxin units/ml and plated into microtiterwells containing cultured L929 cells as is otherwise conventional in thecytolysis assay. In the control, all cells were lysed. Neutralizingantibody was detected by failure of the lymphotoxin to lyse L929 cells.

The animal immunized with glutaraldehyde-polymerized lymphotoxin raisedantibodies which were active in the ELISA assay, but no serumneutralizing activity was detected.

A suspension containing 100 μg lymphotoxin and 1 ml of a 1.64 percentw/v suspension of aluminum hydroxide (Al(OH)₃ ! was prepared and used toimmunize the same mouse. The mouse was injected with 100 μl of thesuspension intramuscularly and 400 μl intraperitoneally. After one weekthe mouse was injected intravenously with 10 μg of unpolymerized andunadsorbed lymphoblastoid lymphotoxin in 100 μl of PBS. A test of a 1/80dilution of the animal's serum three days later indicated the presencelymphotoxin neutralizing antibody.

The spleen from this animal was harvested. 3×10⁷ spleen cells were fusedwith 5×10⁷ murine myeloma cells and plated into microtiter wellscontaining HAT medium and about 3000 peritoneal macrophages/microtiterwell according to the procedure of S. Fazekas De St. Groth, 1980, "J.Immunol. Meth." 35: 1-21. Hybridomas from wells containing supernatantswhich were positive in the above ELISA assay were grown in 1 ml volumeof DMEM medium with 20 percent fetal calf serum, 10 percent NCTC-135medium, 5×10⁻⁵ M beta-mercaptoethanol and HAT, distributed intomicrotiter wells at a statistical average of one cell per well and thencultured in a 1 or 5 ml volume of the same medium. Supernatants werethereafter assayed for neutralizing antibody. Statistically, about 2percent of the ELISA positive hybridomas from the aluminum hydroxideimmunization synthesized neutralizing antibody. High affinitylymphotoxin antibody optionally is selected from this group ofhybridomas.

EXAMPLE 9 Site-Specific Mutagensis of Lymphotoxin

The method of Example 3 is followed exactly in this example except thatsegment 6 of the synthetic oligonucleotide was modified to have thesequence 5'CTCAACTCTGCACCCA3' and its complementary strand (segment 53)modified to have the sequence 3'AGACGTGGGTCGTCGT5'.

The modified oligonucleotides are annealed to the remainingoligonucleotides and ligated into the expression vector as described inExample 6. This vector contains a 2 bp substitution which changed thelysine +28 codon from lysine to histidine. The histidine mutant isexpressed upon transformation of E. coli ATCC 31446.

Other site-directed mutants are prepared in the same fashion, preferablyselecting codons so as to not introduce an EcoRI restriction site thatwould require the use of a partial EcoRI restriction digest in thedigestion of pLTXB1 called for in Example 3. Nor should the mutationsintroduce additional XbaI or BamHI sites into Fragment A (see FIG. 1b),BamHI or HindIII sites into Fragment B or HindIII or BglII sites intoFragment C. Otherwise, partial digestions will be required to properlyassemble the pLTXB1 mutant; digestion to completion would yield adeletion mutant rather than the substitution mutant that is theobjective in this case.

EXAMPLE 10 Identification of Genomic DNA Encoding Murine and BovineLymphotoxin; Amino Acid Sequence of Murine and Bovine Lymphotoxin

The murine and bovine lymphotoxin genes were isolated from genomic-λlibraries. The human lymphotoxin cDNA fragment (PvuII-EcoRI, 600bp) wasradiolabeled with ³² p by nick translation and used as a probe to screena murine genomic DNA-λ library (M600 strain murine genomic DNA inλCharon4A, T. Maniatis et al., Molecular Cloning, p. 31, 1982) and,independently, a bovine genomic DNA library (EP 88622A). Hybridizationwas performed at low stringency in 20 percent formamide (Gray andGoeddel "P.N.A.S. USA" 80: 5842-5846 19831!) and filters were washedtwice in an aqueous solution of 0.3M sodium chloride, 0.03M sodiumcitrate and 0.1 percent SDS. Several phage that hybridized with thehuman lymphotoxin probe were plaque purified (T. Maniatis et al., "Cell"15: 687-701 1978!). Phage DNA was prepared (T. Maniatis et al., "Cell"15: 687-701 1978!), digested with restriction endonucleases and analyzedby Southern hybridization. A 3500 bp EcoRI murine DNA fragment and a2200 bp EcoRI bovine DNA fragment each hybridized with the humanlymphotoxin probe. These DNA fragments were subcloned into plasmidpBR322 and then sequenced by the dideoxy chain-termination method (A. J.H. Smith Methods in Enzymology 65: 560-580 1980!). The deduced proteinsequence of murine and bovine lymphotoxin, along with that of humanlymphotoxin for comparison, is presented in FIG. 4.

EXAMPLE 11 Expression of Lymphotoxin in Yeast Under the Control of theADH Promoter

Plasmid pLTtrp1 is digested with Xba1 in order to open the plasmid atthe Xba1 site just proximal to the lymphotoxin start codon. The Xba1cohesive terminii are blunted by the Klenow fragment of E. coli DNApolymerase I with four dNTPs. An EcoR1 adaptor ##STR1## is ligated tothe blunted plasmid fragment, the protruding 5' hydroxyl terminiiphosphorylated using polynucleotide kinase, the ligation mixture used totransform E. coli ATCC 31446 and a plasmid pLTtrp1R1 identified byrestriction analysis that contains an additional EcoR1 site proximal tothe lymphotoxin start codon. Plasmid pLTtrp1R1 is isolated, digestedwith EcoR1 and the lymphotoxin DNA-containing fragment SP recovered.

Plasmid pFRPn (EP 60,057A) is digested with EcoR1, treated with alkalinephosphatase to prevent recircularization, ligated to the SP lymphotoxinfragment using T4 DNA ligase and the ligation mixture then used totransform E. coli ATCC 31,446. Ampicillin resistant colonies yield twoseries of plasmids having the SP insert in opposite orientations asdetermined by restriction analysis on agarose electrophoresis gels.Plasmids are purified from E. coli transformants and used to transformyeast having the trp1 mutation (for example yeast strain RH218,unrestricted ATCC deposit No. 44076) to the trp⁺ phenotype. Plasmidsoriented such that the start codon of segment SP is located adjacent tothe alcohol dehydrogenase promoter fragment are found to transform theyeast to lymphotoxin expression. Lymphotoxin is recovered from extractsof the yeast transformants. The plasmid stability in large scalefermentations can be improved by employing an expression plasmidcontaining the 2 micron origin of replication in place of the pFRPnchromosomal origin of replication (ars 1) and a compatible host strain(J. Beggs, 1978, "Nature" 275: 104-109).

EXAMPLE 12 Expression of Lymphotoxin in Mammalian Cells

λLT11 (Example 2) is digested with EcoR1 and the lymphotoxin-containingDNA fragment (the reverse transcript) recovered. Plasmid pEHER (EP117,060A) is digested with EcoR1, treated with calf intestinal alkalinephosphatase, and ligated to the EcoR1-linkered reverse transcript ofλLT11. The resulting plasmids grown on E. coli ATCC 31446 (EP 117,060A)and designated pEHERLT I and pEHERLT II. They contained the lymphotoxinDNA in opposite orientations as determined by restriction analysis onpolyacrylamide gels. These plasmids are used to transfect and select CHODHFR-DUX-B11, CHO 1 and Ltk⁻ cells.

Tissue culture cells are transfected by mixing 1 μg of pEHERLT I orpEHERLT II as prepared above with 10 μg rat carrier DNA in a volume of250 μl, 0.25M CaCl₂, followed by dropwise addition of 250 μl HEPESbuffered saline (280 mM MaCl, 1.5 mM Na₂ PO₄, 50 mM HEPES, pH 7.1).After 30 minutes at room temperature, the solution is added to tissueculture cells growing in 60 mm plastic tissue culture dishes. CHO 1, CHODHFR-DUX-B11, and Ltk⁻ cells are used. The dishes contain 3 ml culturemedium appropriate to the host cell.

For CHO 1 and CHO DHFR-DUX-B11 cells, the medium is Ham F-12 media(Gibco) supplemented with 10 percent calf serum, 100 μu/ml penicillin100 μg/ml streptomycin, and 2 μmM L-glutamine. For the Ltk⁻ cell line,the medium is Dulbecco modified Eagle's medium (DMEM) supplemented asabove.

After 3-16 hours, the medium is removed and the cells are washed with 20percent glycerol in phosphate buffered saline. Fresh medium is added toeach plate and the cells are incubated for 2 more days.

Selection of transfected host cells is carried out by trypsinizing thecells after 2 days growth (which comprises treating the cells withsterile trypsin 0.5 mg/ml containing 0.2 mg/ml EDTA) and adding about3×10⁵ cells to 10 mm tissue culture plates with selective media. Fordhfr⁻ cells the medium is a formulation of (F-12 GIBCO) medium lackingglycine, hypoxanthine, and thymidine (GHT⁻ medium). For DHFR⁺ hostcells, methotrexate (100-1000 nM) is added to the normal growth medium.Controls are run using transfection conditions with no plasmid and withplasmid pFD-11 (EP 117,060A) containing normal DHFR. Colonies arisingfrom cells which take up and express the DHFR plasmid are apparentwithin 1-2 weeks. Transformants are identified that express maturelymphotoxin.

We claim:
 1. An isolated DNA molecule comprising(a) the nucleotidesequence of FIG. 2A which encodes amino acid residues his+24 to leu+171as shown in FIG. 2A, or (b) a fragment of the nucleotide sequence of acDNA molecule isolated from a mammalian library which encodes a mature,biologically active mammalian lymphotoxin polypeptide, wherein the cDNAmolecule hybridizes under low stringency conditions to a probe havingthe nucleotide sequence of (a).
 2. An isolated DNA moleculecomprising(a) the nucleotide sequence of FIG. 2A which encodes aminoacid residues leu+1 to leu+171 as shown in FIG. 2A, or (b) thenucleotide sequence of a cDNA molecule isolated from a mammalianlibrary, wherein the cDNA molecule hybridizes under low stringencyconditions to a probe having the nucleotide sequence of (a) and whichencodes a biologically active lymphotoxin polypeptide.
 3. A vectorcomprising the DNA of claim 1 or claim
 2. 4. A host cell comprising thevector of claim
 3. 5. A host cell according to claim 4, wherein the hostcell is an E. coli cell.
 6. A host cell according to claim 4, whereinthe host cell is a yeast cell.
 7. A host cell according to claim 4,wherein the host cell is a mammalian cell.
 8. A method of producing alymphotoxin polypeptide comprising the steps of:culturing a host cellaccording to claim 4 under conditions suitable for expression of thelymphotoxin-encoding DNA therein; and recovering the polypeptide.
 9. Anisolated DNA molecule comprising the sequence shown as nucleotides 1 to1337 of FIG. 2A.
 10. An isolated DNA molecule comprising the nucleotidesequence of FIG. 2A which encodes amino acid residues met-34 to leu+171of FIG. 2A.
 11. An isolated DNA molecule comprising the nucleotidesequence of FIG. 2A which encodes amino acid residues leu+1 to leu+171of FIG. 2A.
 12. An isolated DNA molecule comprising the nucleotidesequence of FIG. 2A which encodes amino acid residues his+24 to leu+171of FIG. 2A.
 13. An isolated variant lymphotoxin having a mutated aminoacid sequence which differs from the native sequence lymphotoxin shownin FIG. 2A, wherein the differences between said mutated and nativesequences consist of one or more amino acid residue deletions,substitutions, or insertions into the sequence of FIG. 2A selected fromthe group consisting of:(a) residues -34 to -1 have been deleted andala-lys is inserted between glu+127 and pro+128; (b) residues -34 to -1have been deleted and ala-lys is inserted between thr+163 and val+164;(c) residues -34 to -1 have been deleted and histidyl is substituted forlysyl+89; (d) residues -34 to -1 have been deleted and valyl, isoleucyl,or leucyl is substituted for alanyl+168; (e) residues -34 to -1 havebeen deleted and tyrosyl is substituted for threonyl+163; (f) residues-34 to -1 have been deleted and lysyl is substituted for seryl+82; (g)residues -34 to -1 have been deleted and isoleucyl, leucyl,phenylalanyl, valyl, or histidyl is substituted for seryl+42; (h)residues -34 to -1 have been deleted and glutamyl, tryptophanyl, seryl,or histidyl is substituted for lysyl+84; (i) residues -34 to -1 havebeen deleted and aspartyl or lysyl is substituted for threonyl+163; (j)residues -34 to -1 have been deleted and lysyl or glycyl is substitutedfor seryl+70; (k) residues -34 to -1 have been deleted and tyrosyl issubstituted for threonyl+69; (l) residues -34 to -1 have been deletedand arginyl or histidyl is substituted for lysyl+28; (m) residues -34 to-1 have been deleted and arginyl or lysyl is substituted forhistidyl+32; (n) residues -34 to -1 have been deleted and prolyl, seryl,threonyl, tyrosyl, or glutamyl is substituted for aspartyl+36; (o)residues -34 to -1 have been deleted and tyrosyl, methionyl, or glutamylis substituted for seryl+38; (p) residues -34 to -1 have been deletedand threonyl, tyrosyl, histidyl, or lysyl is substituted for seryl+61;(q) residues -34 to -1 have been deleted and aspartyl, seryl, or tyrosylis substituted for glycyl+124; (r) residues -34 to -1 have been deletedand arginyl, lysyl, tyrosyl, tryptophanyl, or prolyl is substituted forhistidyl+135; (s) residues -34 to -1 have been deleted and aspartyl issubstituted for threonyl+142; (t) residues -34 to -1 have been deletedand lysyl or threonyl is substituted for glutamyl+146; (u) residues -34to -1 have been deleted and histidyl is substituted for lysyl+19; (v)residues -34 to -1 have been deleted and histidyl is substituted forarginyl+15; (w) residues -34 to -1 have been deleted and one amino acidhas been deleted from the sequence of residues +24 to +171; and (x)residues -34 to -1 have been deleted and one amino acid has beeninserted in the sequence of residues +24 to +171.
 14. An isolated DNAmolecule comprising a nucleotide sequence encoding the variantlymphotoxin of claim
 13. 15. A vector comprising the DNA of claim 14.16. A host cell comprising the vector of claim
 15. 17. A host cellaccording to claim 16, wherein the host cell is an E. coli cell.
 18. Ahost cell according to claim 16, wherein the host cell is a yeast cell.19. A host cell according to claim 16, wherein the host cell is amammalian cell.
 20. A method of producing a variant lymphotoxinpolypeptide comprising the steps of:culturing a host cell according toclaim 16 under conditions suitable for expression of the lymphotoxinvariant-encoding DNA therein; and recovering the polypeptide.